Unstructured intermediate states in single protein force experiments

TL;DR

This study uses models and force experiments to analyze intermediate states in protein folding, revealing structural features and proposing modifications to identify and characterize these states with high precision.

Contribution

It introduces a method to determine the structure and location of intermediate states in protein folding using force experiments and specific modifications.

Findings

Intermediate state I has a core stabilized by native contacts and an extended chain.

Force modifications can reveal the presence and position of the core.

Proposed protocols can distinguish on- and off-pathway intermediates.

Abstract

Recent single-molecule force measurements on single-domain proteins have highlighted a three-state folding mechanism where a stabilized intermediate state (I) is observed on the folding trajectory between the stretched state and the native state. Here we investigate on-lattice protein-like heteropolymer models that lead to a three-state mechanism and show that force experiments can be useful to determine the structure of I. We have mostly found that I is composed of a core stabilized by a high number of native contacts, plus an unstructured extended chain. The lifetime of I is shown to be sensitive to modifications of the protein that spoil the core. We then propose three types of modifications--point mutations, cuts, and circular permutations--aiming at: (1) confirming the presence of the core and (2) determining its location, within one amino acid accuracy, along the polypeptide chain. We also propose force jump protocols aiming to probe the on/off-pathway nature of I.